Adjuvanted nanoparticulate influenza vaccine

ABSTRACT

Vaccine compositions comprising influenza antigens formulated as nanoparticulate water in oil miniemulsions. The vaccines may be formulated at the point of use and are useful in emergency response conditions.

This application claims priority to U.S. Provisional Application No.61/285,332, filed Nov. 5, 2009, the contents of which are incorporatedherein by reference.

TECHNICAL FIELD

The field relates to compositions involving prophylactic nanoparticulateinfluenza vaccines. The field further relates to natural adjuvants usedin conjunction with prophylactic nanoparticulate influenza vaccines. Thefield also relates to point of use kits that comprise prophylacticnanoparticulate influenza vaccines and natural adjuvants. As well, thefield also relates to methods of using said prophylactic nanoparticulateinfluenza vaccines that comprise natural adjuvants.

BACKGROUND

Seasonal influenza constitutes a significant healthcare problem in theU.S., with an estimated 17 to 50 million persons affected yearly.Influenza was responsible for an average of 36,000 deaths annuallythroughout the decade 1990-1999, and up to 1 million deaths worldwide.Elderly patients are at particular risk, accounting for 80-90% ofinfluenza related deaths. In the US, the economic cost of the annual fluseason is estimated at $71-167 billion (WHO, 2003). Moreover, pandemicinfluenza, caused by strains of virus that have undergone antigenicshift to produce a new strain against which there is no pre-existingimmunity in the human population, can have a catastrophic impact onsociety. There were three such pandemics in the 20^(th) century (1918,1957, 1968). Over 20 million deaths worldwide were attributed to thepandemic that began in 1918.

The principal means of preventing influenza and of reducing itscomplications is by vaccination (Nichol, K. L. and Treanor, J. J.,2006). The seasonal influenza vaccine is composed of three strains ofvirus, comprising one representative strain from each of the principalviral types predominantly responsible for annual global influenzaoutbreaks since 1977, including A (H1N1), A (H3N2) and B. There are twoclasses of influenza vaccine, including trivalent inactivated vaccine(TIV), given by intramuscular (IM) injection to individuals aged 6months and older, and live attenuated influenza virus vaccine (LAIV),administered intranasally in healthy, non-pregnant persons aged 2-49(ACIP, 2008/9).

Annual adjustments to the composition of the vaccines are necessary toprovide protection against seasonal variances in antigenic structure dueto viral antigenic drift. Vaccine composition is based on predictions ofwhich strains are likely to be the most prevalent in the next fluseason. Occasionally, a different strain circulates than those includedin the vaccine, resulting in an increased risk of vaccinees contractinginfluenza. Such was the case in the 2003-4 flu season in the U.S., wheninfluenza strains related to the prototypical A/Fujuan/411/2002 (H3N2)were circulating whereas the vaccine contained the partly cross-reactiveA/Panama/2007/99 (H3N2) viral antigens (Treanor, J., 2004). The currentseasonal influenza vaccines provide partial to no cross protectionagainst viral strains not included in the vaccine. A vaccine formulationwith greater potency, which was capable of eliciting antibodiescross-reactive against antigenically dissimilar vital strains, and/oragainst less antigenic epitopes shared by different viral strains, wouldhelp compensate for these shortcomings in specificity associated withthe present vaccines.

Patient age affects the level of protection obtained from vaccinationwith TIV (LAIV is not approved for use in the elderly). In adultsyounger than 65 years of age who are inoculated with TIV that is anantigenic match to circulating virus, clinical efficacy levels of 70-90%are obtained (Podda, A., 2001 and Demicheli. V., 2007). However, inadults older than 65, the TIV clinical efficacy rate drops considerably,to an estimated 30-40% (Goodwin, K., et al., 2006). This has beenattributed to the failure of TIV to significantly reduce infection ratesin the elderly, probably as a consequence of lower response rates andantibody titers in that age group (Betts, R. F. and Treanor, J. J.,2000). Age related decline in the immune system underlies the reducedimmune response to influenza vaccines. However, active immunizationremains the most effective measure to prevent influenza and itscomplications in the elderly. A vaccine that increases efficacy in theelderly, and otherwise immunocompromized individuals, would therefore beof significant benefit to elderly and immunocompromized individuals andsociety at large.

At present, about 425 million doses of TIV are produced annually,although this is still only sufficient to immunize 7% of the world'spopulation (Layne et al., 2009). An additional drawback of the presentvaccine is that shortfalls in production can result in insufficientsupplies of vaccine, potentially leaving at-risk patients unprotected.Such occurred in the 2004-5 influenza season, following the discovery ofcontamination in vaccine lots made at a key manufacturing facility inLiverpool, UK., and subsequent destruction of vaccine lots produced atthat facility. Although, shortcomings at the Liverpool facility havebeen corrected and additional manufacturing sites are underconstruction, there is still potential for shortfalls in the future.Moreover, there may not be sufficient time to produce enough vaccine fornewly emergent strains. This would be particularly serious in theinstance of virus emanating from antigenic shift, which has thepotential to cause a pandemic. Influenza vaccine with enhancedimmunopotency could enable a reduction in the quantity of influenzaantigen on a per dose basis, especially in vaccine administered to younghealthy adults, who typically mount a vigorous response to vaccination.Such dose-sparing capability would help ensure adequate vaccine supplyto protect the population in the event of antigen shortages.Pre-positioned, “point-of-use” presentations of suitable adjuvant wouldfurther extend utility of the vaccine.

A vaccine that increases efficacy in the elderly, or otherwiseimmunocompromized, would be of significant benefit. Moreover, theenhanced immunopotency could induce antibodies against antigenicallydissimilar viral strains, and/or against less dominant epitopes, therebyincreasing the potential for cross-immunoprotection against otherstrains of influenza virus not included in the vaccine. In addition, aninfluenza vaccine with greater immunopotency than the existing vaccineswould have a significant beneficial impact where vaccine supplies arescarce since it could result in a dose-sparing effect, which wouldstretch vaccine stocks to help ensure sufficient supplies to protect thelarge populations.

The development of adjuvanted vaccines represents a promising strategyenhance immunogenicity and thereby overcome the limitations of influenzavaccine for protection of the elderly, vaccine scarcities, andcross-protection against alternative strains of influenza virus.Recognition of these potential benefits has led to extensive effortsdirected at developing adjuvants acceptable for use in man (Palese, T.,2006), including assessments of adjuvanted influenza vaccine in humanclinical trials.

The first strong adjuvant to undergo extensive human testing with viralvaccines was Incomplete Freund's Adjuvant (IFA) (Hilleman, M. R., 1968).Freund's adjuvant is the best known of a general class of adjuvantscomprising an oily phase that is emulsified with an antigen-bearingaqueous phase. IFA consists of 90% purified light mineral oil combinedwith 10% mannide monooleate an emulsifier. IFA is formulated in equalvolume with aqueous phase, containing antigen, to create a 1:1water-in-oil emulsion that constitutes the vaccine. (A more potent form,Freund's Complete Adjuvant, additionally contains 0.5 mg heat killedMycobacterium tuberculosis or butyricum per mL of oily phase, whichsignificantly enhances immunopotency but which is too reactogenic foruse in man.) IFA substantially increases humoral responses toparticulate and soluble antigens in comparison with antigen given inaqueous phase alone. The principal mode of action is believed to resultfrom a depot effect, wherein prolonged release of antigen from theemulsion in the injection site results in sustained stimulation ofantibody production (Freund, J., 1956; McKinney, R. W. and Devenport F.M, 1961; Herbert, W. J., 1968). Additional potential mechanisms havealso been identified, including facilitation of antigen dissemination(as emulsion) via the lymphatic system to distant sites and enhancementof monocyte infiltration to sites of emulsion deposition.

Inactivated influenza vaccine adjuvanted with IFA was tested in humansstarting in the early 1950's. Promising initial immunopotency resultsled to large scale testing in the U.S. Armed Forces and in Great Britain(Hilleman, M. R., 1968; Davenport, F. M., 1968). It was shown that IFAsignificantly enhanced immunogenicity, providing a dose sparing effectwhile concurrently stimulating sustained antibody production relative tonon-adjuvanted influenza vaccine. However, increased injection sitereactions to the IFA adjuvanted vaccine were observed, particularly inearly trials which used less pure adjuvant components. Injection sitereactions observed with IFA ranged from inflammation, to granulomas, tosterile abscesses and cysts (Gupta, R. K., et al, 1993). Local reactionsarose shortly after injection or presented after a period of time hadelapsed following vaccine administration; with some reactions beingdetectable up to a year after injection (Stuart-Harris, C. H., 1957).Persistence of non-metabolizable mineral oil at the injection site isthought to be involved with sustained reactions. Removal of impuritiesfrom the mineral oil and mannide monooleate components, andadministration intramuscularly, helped to substantially reduce localreactions in large follow-up studies. Thus, it was observed that therate of cyst formation in recipients of IFA influenza vaccine was3-23/10,000 (Davenport, F. M., 1961). Local reactogenicity with viralvaccines containing IFA was not limited to influenza vaccine; thus, in8,497 individuals given IFA poliomyelitis vaccine, the combined rates ofinjection site tenderness, induration or nodule formation was 22/10,000(Cutler, J. C., et al, 1963). Other than local injection site reactions,no other side effects are associated with IFA adjuvanted vaccines. Areport that IFA was linked to oncogenicity in male Swiss mice has provedunfounded; elevated rates of cancer have not been observed in man in anextensive long term (35 year) follow up of 13,545 IFA-influenza vaccinerecipients compared to 18,294 controls (Page, W. F., et al, 1993). Thislong term follow up study also identified that cases of hypersensitivityreported in some vaccinees was linked to penicillin contaminant in theviral vaccine preparation, and local cysts, which were linked toimpurities in the mannide monooleate preparation used in the IFA.Despite the identification of factors responsible for reactogenicity andtheir reduction or removal from the vaccine components, mineral oilcontaining adjuvants have not been widely adopted for use in man (Gupta,R. K., et al, 1993).

The pressing need for adjuvanted influenza vaccines suggests that therisk/benefits associated with IFA should be seriously reevaluated.Evidence for the potential benefits for dose sparing, and improvedimmunogenicity and protection in the elderly by incorporating adjuvantsin influenza vaccine formulations can be drawn from studies evaluatingalum-based adjuvants and oil-in-water adjuvants, even though theseadjuvants are typically not nearly so effective immunostimulants as IFA.

Aluminum salts, including aluminum hydroxide and aluminum phosphate, areeffective at enhancing responses to toxoid antigens in man and have beenassessed as adjuvant for influenza vaccines. A 1958-9 study in the U.K.,found that aluminum phosphate failed to enhance antibody responses toinactivated influenza virus vaccine (Himmelweit, F., 1960). Morerecently, a clinical study with an alum-adjuvanted monovalent influenzawhole virus vaccine showed that doses as low as 1.9 μg offeredprotective immunity compared to non-adjuvanted vaccine containing 15 μgHA per dose (Hehme, N. et al, 2004). However, in other studies alum hasfailed to provide any measurable benefit. Thus, immunization with twoinjections of A/H5N1 antigen in alum given at a 1 month intervalprovided no benefit over non-adjuvanted immunogen in a phase I/IIclinical trial conducted in adults aged 18-49 years (Keital, W. A., etal., 2008). Similarly, a randomized double-blind study in 394 healthyadults found no enhancement of antibody titers in response to twoinjections of alum adjuvanted A/H5N1 vaccine in comparison with vaccinein saline (Bernstein, D. I., et al., 2008). The failure of alum toenhance responses in the healthy adults under 65 years of age in thesestudies sheds considerable doubt on the alum's capacity to be ofsignificant benefit to the elderly.

MF59, an oil-in-water adjuvant approved for use in Europe has beencompared to several non-adjuvanted licensed influenza vaccines in over20 clinical trials. The oil in MF59 is squalene, which is metabolizable.The MF59-adjuvanted subunit vaccine, FLUAD is reportedly moreimmunogenic in the elderly than a conventional subunit vaccine (AGRIPAL)(Gasparini, R., et al, 2001) and an inactivated split influenza vaccine(VAXIGRIP) (Squarcione S., et al, 2003) and induced 1.1 to 1.3-foldhigher antibody titers compared to conventional non-adjuvanted splitinfluenza vaccines (Podda, A., 2001, Li, R., et al., 2008).Reactogenicity was reported to be greater in the FLUAD recipients,although not limiting, with higher rates of mild and transient localreactions being observed. The benefits of the vaccine were considered tooutweigh the drawbacks, and FLUAD is approved for use in Europe.However, FLUAD has not exhibited enhanced efficacy in all patientpopulations. Thus, FLUAD reduced pneumonia related hospital admissionrates by 50% in patients over 64 years of age relative to non-vaccinatedcontrols (Puig-Barbera, J., 2004). However, no advantage was seen withFLUAD in terms of reported rates of influenza symptoms in hearttransplant recipients on immunosuppressive regimens (Magnani, G., et al,2005).

The potential health care benefits of enhancing the potency of influenzavaccine are well recognized and have stimulated considerable research inthis field. Although limited improvements have been demonstrated foradjuvanted influenza vaccines formulated with alum and MF59, thereclearly remains the need for improvement in this area. The presentinvention relates to adjuvanted influenza vaccines consisting of fluantigens formulated in a water-in-oil emulsion comprising naturallyoccurring oils and emulsifiers derived from vegetable sources. IFA, awell known water-in-oil adjuvant system, has already been demonstratedto significantly increase the immunopotency of influenza vaccineantigens. The influenza vaccines of the invention can be manufacturedeither point-of-use allowing pre-positioning of the adjuvant vehicle inthe supply chain or in bulk by a robust, reproducible, and process.

Testing has been conducted with MF59 oil-in-water emulsion adjuvant(Chiron/Novartis) whereby it was demonstrated that MF59 modestlyenhanced ihe immunogenicity of seasonal influenza trivalent subunitvaccine and was accompanied by an acceptable safety profile in theelderly. Mean antibody titers against the three viral strain-specifichemagglutinin components of the vaccine were higher in patientsreceiving MF59 adjuvanted vaccine, with mean serum antibody titer ratiosof adjuvant versus non-adjuvant cohorts ranging from 1.1 to 1.3 fold,depending on the viral component against which the sera were assayed(Gasparini, R., et al, 2001). Even higher response ratios wereassociated with patients with pre-immunization titers ≦40 in a differentstudy (De Denato, S., et al, 1999). These studies reported higher ratesof injection site reactions for patients vaccinated with the MF59adjuvanted formulation than with standard vaccine, though the majorityof such reactions were mild and the difference in reaction rates was notfound to be significant relative to the reactogenicity of non-adjuvantedvaccine. The results of the clinical trials, summarized by Podda, A.,2001) were sufficiently favorable that the MF59 influenza vaccine(Fluad™) was granted approval in Europe. The experience with MF59demonstrates that split virion vaccine can be formulated with adjuvantwithout adversely affecting performance as assessed by serologicalassays for anti-influenza antibody titers and standard safetyparameters.

SUMMARY OF THE INVENTION

This invention involves prophylactic influenza vaccines comprisinginfluenza vaccine antigens formulated as a nanoparticulate water-in-oilemulsion with an adjuvant vehicle derived from naturally occurring oilsand emulsifiers. The invention is advantageous, in that it can beformulated by a batch manufacturing process (BMP) on a large scaleadequate for prophylactic vaccines, or it can be formulated at the pointof administration with oil adjuvant vehicle and influenza vaccineantigens as a miniemulsion by a point-of-use (POU) mixing method.

The vaccine of the invention, depending on the influenza antigens usedin its formulation, should induce the production of immunoprotectiveantibodies against antigenically dissimilar virus strains and/or againstadditional less antigenic epitopes that cross react between viralstrains to a much greater degree than is characteristic for antibodiesinduced by the standard, non-adjuvanted vaccine and should extend theperiod after immunization that immunoprotective titers remain elevated.The vaccine compositions comprise nanoparticulate water-in-oilemulsions. The aqueous phase of the emulsions comprises from about 25%to about 35% by weight of the composition and contains the influenzaantigen or antigens. The oil phase of the emulsions comprises anadjuvant oil comprising squalene and squalane, a first emulsifier whichis mannide monooleate or sorbitol monooleate and a second emulsifierselected from the group consisting of polyoxyl-40-hydrogenated castoroil, sorbitan monopalmitate, a polysorbate (e.g Tweens), Hypermer B239or Hypermer B246. The nanoparticulate emulsions are formulated so thatthe median aqueous globule size is from about 0.3 μm to about 1 μm.

One example of the invention concerns a method for the point of useformulation of adjuvanted nanoparticulate water-in-oil emulsionvaccines. The method comprises preparing an aqueous solution of aproteinaceous antigen such as an influenza antigen, transferring asuitable amount of the aqueous solution to a sterile containercontaining a predetermined amount of the adjuvant oil, shaking thecontainer so as to mix the contents to form a milky pre-emulsion, andtransferring an aliquot of the milky pre-emulsion to a first sterilesyringe. The first sterile syringe is connected to a sterile three-waystopcock to which a second sterile syringe is attached at a 90 degreeangle to the first syringe. The emulsion is passed from one syringe tothe other by manually depressing the plunger of each respective syringein a serial fashion for a sufficient number of cycles so as to create anemulsion of the aqueous solution in the adjuvant oil wherein the medianglobule size is from about 0.3 μm to about 1 μm.

As a result of the enhanced immunogenicity, the inventive vaccinesshould enable a reduction in the quantity of antigen delivered per doseof vaccine, thereby extending antigen stocks to provide for theproduction of larger quantities of vaccine from the same quantity ofantigen. This would be of particular benefit for both the alleviation ofshortfalls in annual manufacture of vaccine as well as enabling anincrease in the number of doses of vaccine in limited supply to counternewly emerging influenza strains, such as avian influenza and pandemicstrains.

The “point of use” vaccine products permit pre-positioning of theadjuvant vehicle for local, as needed formulation with the thenprevailing influenza strain viral antigens, and should result insignificant supply chain and potential cost savings benefits to nationalgovernments planning pandemic flu preparedness. The adjuvant is suitablefor combination with influenza vaccine preparations that targetdifferent strains of virus and thus, can be used with vaccines that areadjusted for viral specificity on an annual basis. An added benefit ofthe vaccines of the invention should be to increase influenza vaccineresponse rates in the elderly and otherwise immunocompromised subjects.

DESCRIPTION OF THE FIGURES

FIG. 1: Comparison of POU and BMP Vaccine Emulsions according to theinvention (MAS-1). Mean globule size distribution D(v,0.5)±SEM of“point-of-use” emulsions of as little as 1.0 mL are within specificationof ≦1 μm, and comparable to the emulsions produced by the bulkmanufacturing process at 0.4, 2.0 and 10 L. Protein antigen (solid bars)up to at least 9.25 mg/mL in the aqueous phase has no effect on globulesize distribution.

FIG. 2: SDS-PAGE of Fluzone, aqueous phase and extracted aqueous phasesamples. Test Samples: Fluzone (1) 15 μg/mL, (2) 5 μg/mL; (3) TIVfiltrate; TIV concentrate (4) 1:3 dilution, (5) 1:27 dilution); TIVconcentrate used in a vaccine according to the invention (MAS-1/TIV) (6)1:3 dilution, (7) 1:9 dilution; MAS-1/TIV aqueous phase extract (8) 1:3dilution, (9) 1:27 dilution. MW standards: Precision Plus ProteinStandards, BioRad #161-0374: mol. Wt. 10, 15, 20, 25, 37, 50, 75, 100,150, 250 KDa. using In Vitrogen Novex 10% Mini-Gel (#7102570-0877) withNuPAGE MOPS SDS running buffer (25 mL+475 mL H₂O).

FIG. 3A: Immunopharmacokinetics, Geometric mean HAI antibody titers tothe viral antigen A/H1N1 in mice (n=6/grp) immunized subcutaneously onday 0 and day 28 with doses of H Ag formulated with MAS-1 (0.05, 0.15,0.4, 1.3 and 4.0 μg) and control TIV (4.0 μg).

FIG. 3B: Immunopharmacokinetics, Geometric mean HAI antibody titers tothe viral antigen A/H3N2 in mice (n=6/grp) immunized subcutaneously onday 0 and day 28 with doses of H Ag formulated with MAS-1 (0.05, 0.15,0.4, 1.3 and 4.0 μg) and control TIV (4.0 μg).

FIG. 3C: Immunopharmacokinetics, Geometric mean HAI antibody titers tothe viral antigen B/Malaysia in mice (n=6/grp) immunized subcutaneouslyon day 0 and day 28 with doses of H Ag formulated with MAS-1 (0.05,0.15, 0.4, 1.3 and 4.0 μg) and control TIV (4.0 μg).

FIG. 3D: Dose Response Analysis, Day 56 Geometric Mean HAI titers to theA/H1N1, A/N3N2 and B/Malaysia viral anitgens.

FIG. 4A: Immunopharmacokinetics, Geometric mean HAI antibody titers tothe viral antigen A/H1N1 in rabbits (n=5/grp) immunized on day 0 and day28 intramuscularly with doses of H Ag formulated with MAS-1 (1.67, 5.0and 15 μg) and control TIV (15 μg).

FIG. 4B: Immunopharmacokinetics, Geometric mean HAI antibody titers tothe viral antigen A/H3N2 in rabbits (n=5/grp) immunized on day 0 and day28 intramuscularly with doses of H Ag formulated with MAS-1 (1.67, 5.0and 15 μg) and control TIV (15 μg).

FIG. 4C: Immunopharmacokinetics, Geometric mean HAI antibody titers tothe viral antigen B/Malaysia in rabbits (n=5/grp) immunized on day 0 andday 28 intramuscularly with doses of H Ag formulated with MAS-1 (1.67,5.0 and 15 μg) and control TIV (15 μg).

DETAILED DESCRIPTION

In one example, the vaccines of the invention can be produced either“point-of use” or as a “bulk filled” final drug product.

As a “point-of-use” product, the vaccine is formulated as ananoparticulate emulsion made by a simple, but robust and reproduciblehand mixing procedure. This is illustrated by the globule size diameter50% distribution results (D(v,0.5) for emulsions determined by laserlight diffraction between multiple operators and their stability at roomtemperature. At T zero, globule size distribution for “point-of-use”emulsions prepared by 4 operators at the lower and upper mixing limitsof the method were N=29; Mean D(v,0.5)±SEM=0.44±0.03 μm and N=26; MeanD(v,0.5)±SEM=0.66±0.03 μm, respectively. Globule size measurements at 2,18-24 and 48 hours after preparation indicated that there was nosignificant change in this parameter over the two-day storage period.

“Bulk-filled” vaccine emulsion is manufactured aseptically and filledinto single use containers such as vials or ampules. The mechanized,bulk fill process should typically produce emulsions with a mean globulesize diameter of approximately 0.30 μm. Protein antigen up to at least9.25 mg/mL in the aqueous phase has no effect on globule sizedistribution for emulsions made by either the point-of-use or the bulkfill method.

The vaccine emulsions have a low viscosity affording syringeability sothat as little as 0.1 mL doses can be administered to patients with highprecision and reproducibility.

The components of the oil adjuvant vehicle suitable for use in theinvention, comprise a first sugar ester emulsifier such as mannidemonooleate (MMO) or sorbitan monooleate, a second emulsifier such as ahydrogenated castor oil, for example, polyoxyl-40-hydrogenated castoroil (POCO), and naturally occurring and metabolizable oils, preferablysqualene and squalane. The metabolizable oils typically comprise fromabout 85% to about 90% by weight of the oil, the first sugar esteremulsifier from about 6% to 15%, i.e., about 9% to about 12%, or about10% or 11% by weight of the oil, and the second emulsifier from about0.1%-1.1%, i.e., 0.2% to about 1%, 0.4 to about 0.8%, 0.5% to about0.7%, or about 0.6% by weight of the oil. The metabolizable oilcomponent may be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% squalene,and 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% squalane by weight,but the concentration of these components may vary plus or minus 10%within this component. A suitable adjuvant vehicle for use in theinvention is MAS-1, which is comprised of naturally occurring andmetabolizable components derived from vegetable sources, and iscommercially available from Mercia Pharma, Inc, Scarsdale, N.Y.(www.merciapharma.com). Point-of-use (POU) vaccines may be produced withthese adjuvant oils using a robust and reproducible hand-mixing methoddescribed herein.

The components of the oil vehicle, including their starting materials,which may be derived from either animal or vegetable sources, orcombinations thereof, are all commercially available from multiplesources. Suitable sugar esters as the first emulsifier in addition toMMO include polysorbates, particularly sorbitan monooleate. In additionto POCO as the second emulsifier sorbitan esters, such as sorbitanmonopalmitate, polysorbates, such as the Tweens family of emulsifiers,and Hypermers B239 and B246 may be useful.

The nanoparticulate vaccine emulsions of the invention typically containfrom about 65% by weight to about 75% by weight of the adjuvant oilvehicle, but may also contain about 10%, 20%, 30%, 40%, 50%, 60%, 70%,80% or 90% by weight of the adjuvant oil vehicle. The nanoparticulatevaccine emulsions of the invention typically contain from about 25% toabout 35% by weight of an aqueous phase containing the protein antigen,but may also contain from about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,or 90% by weight of an aqueous phase containing the protein antigen. Incertain embodiments of the invention the aqueous phase comprises fromabout 27% to about 33% by weight of the vaccine emulsion.

The water-in-oil vaccine emulsions used in the invention should beformulated so that the aqueous globules in the emulsion carrying theantigen have median diameters less than 1 micron with median diametersin the range from about 100 nanometers to about 1 micron, and typicallywith an average diameter of about 300 nanometers. It is furthercontemplated that the aqueous globules may be 50 nanometers, 100nanometers, 150 nanometers, 200 nanometers, 250 nanometers, 300nanometers, 350 nanometers, 400 nanometers, 450 nanometers, 500nanometers, 550 nanometers, 600 nanometers, 650 nanometers, 700nanometers, 750 nanometers, 800 nanometers, 850 nanometers, 900nanometers, or 950 nanometers. The oil components of the adjuvant arepreferably naturally occurring biological oils that are metabolizable,unlike the mineral oil that comprises the oil phase of the well knownFreund's adjuvants (both incomplete and complete formulations).

The vaccine emulsions of the invention should tolerate highconcentrations of antigen, such as from 0.1 mg/mL to 20 mg/mL, i.e, 1mg/mL, 5 mg/mL, 7 mg/mL, 9 mg/mL, 10 mg/mL, 11 mg/mL, 13 mg/mL, 15mg/mL, 20 mg/mL, or up to at least 10 mg/mL and should be compatiblewith commonly used protein solubilizers (e.g., 4M urea, 30% DMSO).Unlike IFA emulsions, they should be compatible with aqueous phaseshaving a wide range of pH, i.e., from about 2-9, 3-8, 4-6, i.e., about5, and unaffected over a wide range salt concentrations. Unlike IFAemulsions (>1,500 cP), the vaccine emulsions of the invention shouldhave a low viscosity (<100 cP) as free flowing emulsions permitting highprecision low volume (0.1 mL) dosing. The physico-chemicalcharacteristics of the vaccine emulsions of the invention should have amedian distribution of globule size diameter of (D(v,0.5)) less than orequal to 1.0 μm, and be unaffected by high concentrations of protein inthe aqueous phase (FIG. 1).

At T zero, D(v,0.5) μm±SEM for POU vaccine emulsions prepared by fourdifferent operators at the lower and upper mixing limits of the methodwere; 0.44±0.30 (n=29) and 0.66±0.03 (n=26), respectively. D(v,0.5)after 2, 24 and 48 hours at ambient temperature indicated no significantchange. At release, BMP vaccine emulsions should have a D,(v0.5) of 0.3μm with an end of shelf life D,(v0.5) of ≦1 μm after 3 years at 2-8° C.without any loss in immunopotency in vivo (by contrast D(v,0.5) of IFAis 3-10 μm a T zero).

The invention is further described in the following examples. Theexamples are merely illustrative and do not in any way limit the scopeof the invention as described and claimed.

EXAMPLE 1

TIV Vaccines: In one example of the invention the vaccines are comprisedof standard TIV antigens derived from egg or cell cultures located inthe aqueous phase, formulated as a 30:70 (w/w/) water-in-oil (w/o)nanoparticulate emulsion with MAS-1 having a median globule sizediameter D(v,0.5) of 0.30 μm. A suitable adjuvant vehicle for use in theinvention is MAS-1.

Formulation of TIV influenza vaccine: Fluzone TIV, was obtained fromSanofi Pasteur, as the source of H antigen (Ag) for these studies.Fluzone contains 30 μg,/mL of each H antigen, and yields 9 μg/mL of eachH antigen formulated in a 30:70 emulsion with MAS-1 adjuvant. FluzoneTIV was concentrated up to 10-fold using Amicon Ultra 15centrifugational concentrators (cut-off: 10,000 kd). Duringconcentration of TIV approximately 50% of total protein determined byLowry assay is recovered in the concentrate. Approx. 40% of totalprotein passes through the filters into the filtrate, and 12% isadsorbed to the ultrafilters (Table 1). TIV protein recovery wasreproducible between batches. Total protein analyses of the aqueousphase by Lowry confirmed batch-to-batch consistency across the range ofH Ag concentrations, with protein content of the aqueous phase varying≦2.5% and 3.5% for Fluzone between batches (Table 2).

TABLE 1 Recovery and Mass Balance of TIV protein during centrifugalconcentration Aqueous Phase Total Protein (μg) Total Protein (μg) TestSample Batch 1 Batch 2 Fluzone 12,020 12,440 Concentrate 5,893 (49.0%)5,928 (47.7%) filtrate 4,662 (38.8%) 5,130 (41.2%) Balance 12.2% 11.1%

Although total protein was lost, H antigen was not lost during theconcentration process. Total H antigen by single radial immunodiffusion(SRID) test comprises 14-15% of the total protein measured by Lowry(against BSA standard) in Fluzone, and 30-31% of the total protein inthe vaccine emulsion formulations (Table 2). The validated SRID assay isused for batch release of approved influenza vaccines. We establishedthe SRID assay in our laboratory, using the WHO methods and referenceantisera and antigens. By SRID, all three H antigens were retained inthe concentrate with no loss into the filtrate. These results wereconfirmed by SDS-PAGE run on samples of concentrate and filtrate (FIG.2).

TABLE 2 Content and reproducibility of H antigen and total protein inMAS-1/TIV Protein % [H Ag]/ Protein % [H Ag]/ Total (μg/mL) by [SRID](μg/mL) by [SRID] Immunogen (μg/mL) Lowry² for for Lowry² for for %(Batch-1/ For Group H Ag¹ Batch 1 Batch 1 Batch 2 Batch 2 Batch-2) 1(Fluzone) 90.0 601 15% 622 14% −3.49% 2 150 496 30% 499 30% −0.60% 3 50165 30% 166 30% −0.61% 4 50 159 31% 163 31% −2.52% 5 150 496 30% 499 30%−0.60% 6 16.7 55 30% 56 30% −1.82% 7 50 159 31% 163 31% −2.52% 8 5.718.3 31% 18.7 30% −2.19% 9 (Placebo) 0 0 NA 0 NA NA ¹Total H Agconcentration from Fluzone specification ²Total protein (BSAequivalents) measured by Lowry against BSA standard; NA Not applicable

EXAMPLE 2

POU Mixing Procedure Parameters: The range of mixing rate and mixingcycles optimal for making water-in-oil miniemulsion nanoparticulatevaccine emulsions according to the invention were established using anaqueous phase comprising phosphate buffered saline (PBS), pH 7.2 andMAS-1 oily vehicle in a 30:70 (w/w) ratio made using the POU mixingprocedure. A range of mixing rates and mixing cycles were evaluated atsyringe sizes from 1.0 to 5.0 mL and emulsion volumes ranging from 1.0to 3.0 mL. Samples were analyzed immediately after preparation (T-zero)and at select times after storage at ambient conditions for up to 1week. The data are presented in Table 3.

TABLE 3 Physico-chemical characteristics of POU 30:70 (w/w) MAS-1Vaccine emulsions Mixing Syringe Size/ Globule size diam, D(v, 0.5)Replicates Mix Duration emulsion vol Mean (SD) μm (N)* Cycles (sec) (mL)T-zero 4-6 hr 24 hr 48 hr 1 week 8 40 No 1/1 7.68 11.37 1.58 nr nrConstraint (10.2) (14.6) 3 50 No 1/1 1.46 2.04 nr nr nr Constraint(0.62) 3 60 No 1/1 12.00 nr 14.29 nr nr Constraint (17.76) (18.00) 6 75No 1/1 1.16 1.40 nr nr nr Constraint (0.32) 1 100 No 1/1 0.62 nr 0.70 nrnr Constraint 11 125 No 1/1 0.64 nr 0.40 nr nr Constraint (0.20) 6 125 90 1/1 0.33 nr 0.36 0.44 0.40 (0.04) (0.11) 20 125 100 1/1 0.47 0.310.43 0.48 0.53 (0.20) (0.11) (0.01) (0.33) 6 125 110 1/1 0.74 nr nr nrnr (0.07) 17 125 120 1/1 0.60 0.57 0.70 0.83 0.82 (0.16) (0.13) (0.16)(0.07) (0.19) 2 125 150 1/1 nr nr 0.91 nr 0.99 (0.07) 3 125  90 3/2 0.310.30 0.31 0.30 nr (0.01) 1 125 180 3/2 0.75 nr nr nr nr 1 125 135 5/50.30 nr 0.31 nr nr Note: nr = not reported; *Replicates (N) refers tothe number of emulsions produced under the stated condition

The combined (N=52) results for physico-chemical characteristics of 1and 2 mL POU MAS-1 vaccine emulsions prepared with 1 and 3 mL syringes,respectively, made by 125 cycles in the range 90 to 120 secondspresented in Table 4.

TABLE 4 Physico-chemical characteristics of POU 30:70 (w/w) MAS-1Vaccine emulsions Storage at Ambient Temperature Characteristics T zero4-6 hr 24 hr 48 hr 1 wk Viscosity cP 86 nr nr nr nr Globule size D(v0.5)0.52 (0.2) 0.44 (0.17 0.47 (0.17) 0.56 (0.22) 0.61 (0.27) Mean (SD) μmAppearance score  0 (0) 0 0 0 nr

Key to Appearance Scores

The samples, contained in clear glass vials, are visually examined underdefined lighting for the presence of aqueous droplets collecting at thebottom of the emulsion. The method gives a macroscopic measure of thequality of the emulsion.

0 No aqueous droplets were observed 1-3 Increasing amounts (quantity andsize) of aqueous droplets were observed 4 An aqueous pool was observed 5An aqueous layer was observed 6 a complete phase separation was observed

The POU method produces reproducible, robust, and stable MAS-1 vaccineemulsions when mixed for 125 cycles within 90-120 seconds at the 1 to 2mL. Scale and 90-150 seconds at the 5 mL scale.

EXAMPLE 3

Immunopotency Enhancement in Mice of MAS-1/TIV: The objective of thisdose ranging study was to evaluate the immunopotency and dose sparingbenefits of MAS-1 adjuvant on TIV in mice. The study was designed togenerate data on primary and secondary response parameters, includingantibody titers, response kinetics, isotype and specificity followingimmunization with the adjuvanted vaccine of the invention compared tostandard TIV vaccine. General well-being of the animals was monitoredthroughout the study period.

Test formulations: Prior to emulsification with MAS-1 vehicle, H antigenfrom TIV concentrate was diluted with PBS to yield aqueous phases withthe correct concentrations of H antigen. H antigen concentrations werecalculated from the H antigen content of the starting material (Fluzone)and the volume following concentration by centrifugal ultrafiltration.The aqueous phase protein contents were confirmed by Modified LowryAssay (Pierce Chemical Co.). The TIV aqueous phase preparations werereadily emulsified in MAS-1 oil phase vehicle using the POU mixingprocedure.

The vaccine emulsions were broken to extract the aqueous phase and oilphases. SDS-PAGE analysis of antigen-bearing aqueous phase extractedfrom the emulsion demonstrated that the antigens are compatible with theMAS-1 adjuvant. Protein mobility and band numbers were the same betweenaqueous phase and extracted aqueous phase.

Study outline: The study was a seven-arm, placebo controlled study atdoses/H antigen (A/H1N1; A/H3N2; and, B) of 4.0 μg as TIV (+control);MAS-1 placebo (−control), and 0.05 to 4.0 μg of TIV formulated as ananoparticulate emulsion with MAS-1 according to the invention. BALB/cmice (female, 8-12 weeks old at start of study, 6 per group) wereimmunized with 0.1 mL test articles subcutaneously (dorsal, base ofneck) on days 0 and 28. Blood samples were taken on days 0, 14, 28, 42,and 56. Sera were prepared and stored at −20° C. until assay. Theformulations were prepared from clinical grade 2007/08 season TIV(Fluzone). Fluzone was concentrated 10-fold to 452 μg/mL total H antigenaqueous phase. The vaccine and placebo emulsions were prepared by thePOU method. The aqueous phase protein content for the vaccinepreparations were verified by Modified Lowry and SDS-PAGE.

General Safety: None of the mice showed any signs of distress or illhealth over the course of the study at any of the doses testedindicating that the formulations were safe and well tolerated.

Immunopotency and enhancement: The hemagglutination inhibition (HAI)titers against each of the three viral strains used in the 2007/08 TIV(A/Solomon Islands/3/2006 (H1N1); A/Wisconsin/67/2005 (H3N2);B/Malaysia/2506/200) were measured using the WHO assay protocol andreference standards. Testing was performed blinded to group assignment.Group geometric mean HAI titers at days 42 and 56 for the threeinfluenza viral antigens and the dose adjusted enhancement of the immuneresponse in MAS-1/TIV vaccine according to the invention for eachantigen relative to TIV (Group 1) are presented in Table 5. MAS-1significantly enhanced the immunopotency of TIV against all threecomponent viral strains at both time points, particularly on day 56. Atthe matched 4 μg dose, MAS-1 enhanced the day 56 responses by 9 fold forA/H1N1 virus, 11.3 fold for A/H3N2 virus, and 6.3 fold for B virus.Higher HAI titers were observed at day 56 even at 0.15 μg/H antigen inthe adjuvanted vaccine compared with 4 μg/H antigen in standard TIVgroup 1, with dose adjusted enhancement of 48-fold for A/H1N1, 85-foldfor A/H3N2, and 23-fold for B/Malaysia. Overall, dose-adjustedenhancement of TIV in MAS-1/TIV at day 56 compared to standard TIV was22 fold for A/H1N1, 26-fold for A/H3N2, and 9-fold for B/Malaysia.

TABLE 5 Geometric mean HAI titers of test groups and dose-adjustedenhancement of HAI titers relative to Fluzone positive control (group1). Titer A/H1N1 A/H3N2 B Bleed day Group Treatment 42 56 42 56 42 56 1TIV 4 μg Geo. Mean 1,280 905 1,611 905 285 226 (+) Control — — — — — — 2MAS-1/TIV 4 μg Geo. Mean 5,747 8,127 25,803 10,240 905 1,437 Enhanced4.5 9.0 16.0 11.3 3.2 6.3 3 MAS-1/TIV 1.3 μg Geo. Mean 2,874 4,561 9052,032 137 359 Enhanced 6.8 15.2 1.7 6.8 1.4 4.8 4 MAS-1/TIV 0.4 μg Geo.Mean 1,016 1,613 118 149 9 16 Enhanced 7.2 16.2 0.7 1.5 0.3 0.6 5MAS-1/TIV 0.15 μg Geo. Mean 806 1,613 1.016 2,874 20 194 Enhanced 16.847.5 16.8 84.7 1.9 22.9 6 MAS-1/TIV 0.05 μg Geo. Mean 5 7 3 3 1 1Enhanced 0.03 0.6 0.1 0.3 0.3 0.4 7 MAS-1 placebo Geo. Mean 1 1 1 1 1 1(−) Control — — — — — — Combo 0.15-4.0 Enhanced 8.8 22.0 8.8 26.1 1.78.7 Note: Enhancement = (test group mean/group 1 mean) × (H Ag dose ofgroup 1/H Ag dose of test group)

Immunopharmacokinetics: Antibody response kinetics were similar for eachof the three component strains (FIGS. 3.A-C). Predictably in naïve mice,relatively low HAI responses were seen following injection 1 in allresponding groups, while enhanced responses followed injection 2,indicative of immune priming by the first dose. By day, 56 HAI titerswith TIV were beginning to fall off, whereas for the MAS-1 adjuvantedvaccine, HAI titers were still rising, indicating that immunization withthe adjuvanted vaccine of the invention extends the period for providingprotection over standard TIV. The relative HAI titers for B viruselicited by TIV were significantly lower than for A virus strains,whereas, in the adjuvanted vaccine, B virus titers were comparable to Avirus titers in standard TIV.

Dose response: The dose-related response of HAI titers for each of thethree viral antigens, A/H1N1, A/H3N2, and B virus elicited by theadjuvanted formulations of the invention is further illustrated by thedata for day 56 presented in FIG. 3D. These data show that 0.15 to 4μg/H antigen in MAS-1 is in a titrating range, and indicate that themouse model should be suitable as ati in vivo immunopotency release andstability indicating assay. Precision will be attained by using 10 up to25 animals per group with release specifications to be made relative toTIV control.

EXAMPLE 4

Evaluation of Efficacy and Safety in Rabbits

Study outline: A dose ranging, placebo controlled 9 arm (N=5/group)study was performed in rabbits to evaluate enhancement of immunopotency,dose sparing potential, and duration of immunoprotective response versusstandard TIV at doses and route of administration anticipated in humans.Additionally, the study incorporated both general safety and a formalevaluation of toxicology to evaluate injection site tolerance andsystemic safety by necropsy and histopathology on a panel of organs. Thestudy encompassed a range of variables, including: MAS-1 vs. no adjuvant(Fluzone), dose of H antigen in MAS-1 (0, 0.56, 1.67, 5.0, and 15.0 μg/Hantigen), injection volume (0.1 and 0.3 mL), and formulation strategy(emulsified to strength or dilution of vaccine emulsion to H antigenstrength with placebo emulsion). Animals were immunized intramuscularlyon days 0 and 28. Blood samples were taken on day 0 and at 2 weeklyintervals through day 84. Sera was prepared from the blood samples andstored at −20° C. until assay. Animals were sacrificed on day 84,necropsied and organs collected for gross evaluation and histopathologyin compliance with GLP. Formulations were prepared from clinical grade2007/08 season TIV (Fluzone), and vaccine and MAS-1 placebo emulsionswere prepared by the POU method. The aqueous phase protein content forvaccine preparations was verified by Modified Lowry and SDS-PAGE.

Immunopotency and enhancement: HAI titers were measured against thethree viral strains present in the 2007/08 TIV, and against the threeantigenically dissimilar strains in the 2008/09 TIV to evaluatecross-immunoprotection. Testing was performed blinded to groupassignment. Peak HAI titers for the three 2007/08 and 2008/09 seasonvirus strains and dose adjusted enhancement of HAI titers by MAS-1 foreach antigen are presented in Tables 6A and B, respectively. Theimmunopharmacokinetics of the immune response induced by TIV andMAS-1/TIV after IM immunization are shown in FIGS. 4A, B and C. MAS-1significantly enhanced the immunopotency of TIV antigens at all dosesagainst 2007/08 viral strains compared to TIV (positive control—group1). At the matched 15 μg dose, MAS-1 enhanced peak responses 3-fold forA/H1N1, 3.5-fold for A/H3N2, and 2-fold for B viruses. Even at 0.56 μg/Hantigen, MAS-1 enhanced HAI titers compared with 15 μ/H antigen instandard TIV by 2-fold for A/H1N1, 1.7-fold for A/H3N2, and 1.3-fold forB viruses, with dose adjusted enhancement of 54-fold for A/H1N1, 47-foldfor A/H3N2, and 35-fold for B/Malaysia. ANOVA comparisons between HAItiters for each of groups 3, 4 and 5, at 5 μg H antigen, and groups 6and 7 at 1.67 μg/H antigen were statistically equivalent, indicatingthat differences in dose volume of 0.1 vs. 0.3 mL, or in formulationstrategy (direct emulsion vs. diluted emulsion) were equivalent. Group9, comprising negative controls injected with placebo MAS-1 emulsion,produced no detectable anti-influenza antibodies (Table 6A).

TABLE 6A Peak Geometric mean HAI titers and dose adjusted enhancement ofMAS-1/TIV Against 2007/08 Season Virus Strains A/H1N1 A/H3N2 B 2007/08Solomon Islands/3/2006 Wisconsin/67/2005 Malaysia/2506/2004 Season DoseVol p p p Grp (μg) (mL) Titer Enhanc value Titer Enhanc value TiterEnhanc value 1 15 0.5 2941 — — 3378 — — 2941 — — 2^(a) 15 0.3 8914 3.00.001 11763 3.5 0.015 5881 2.0 0.059 3^(b) 5 0.3 7760 7.9 0.005 7760 6.90.086 5881 6.0 0.089 4^(a) 5 0.3 11763 12.0 0.018 11763 10.4 0.004 89149.1 0.002 5^(c) 5 0.1 4457 4.5 0.207 6756 6.0 0.034 6756 6.9 0.031 6^(a)1.67 0.3 3881 11.9 0.326 4457 11.9 0.242 2561 7.8 0.620 7^(a) 1.67 0.14458 13.6 0.167 8914 23.7 0.203 3379 10.3 0.658 8^(b) 0.56 0.3 5882 53.60.030 5881 46.6 0.066 3882 35.4 0.460 9 0 0.3 0 — — 0 — — 0 — — Overall— — — 15.21 — — 15.57 11.07 ^(a)Emulsified directly to the indicatedconcentration of H antigen. ^(b)Emulsion prepared at strength for Group2 was diluted 1:3 with placebo emulsion ^(c)Same emulsion as that usedfor Group 2. Enhancement over TIV = (test group mean/group 1 mean) × (HAg dose of group 1/H Ag dose of test group)

EXAMPLE 5

Dose response: The dose response for the adjuvanted vaccine seen in miceappeared to be in the linear range. By contrast, the dose response ofHAI titers for each of the three viral antigens, A/H1N1, A/H3N2, and Bvirus elicited after IM immunization in rabbits indicate that the lowestdose tested, 0.56 μg/H antigen, in MAS-1/TIV is close to the plateauresponse above the dose titration range—lower doses were not evaluated.

Cross-immunoprotection against 2008/09 Season Strains: Immunization witha vaccine according to the invention (MAS-1/TIV, using 2007/2008 TIV)was significantly more cross protective than standard TIV with HAItiters against 2008/09 TIV strains. At the 5 μg/H antigen dose all threeMAS-1/TIV preparations were statistically equivalent by ANOVA, andenhanced on average 4.1-fold for A/H5N1, 3.4-fold for A/H1N1, and2.4-fold for B viruses. The mean dose adjusted enhancement at 5 μg/Hantigen was 12-fold for A/H1N1, 10-fold for A/H3N2, and 7-fold forB/Florida (Table 6B).

TABLE 6B Day 56 Geometric mean HAI titers and dose adjusted enhancementof MAS- 1/TIV Against 2008/09 Season Virus Strains A/H1N1 A/H3N2 B2007/08 Brisbane/59/2007 Brisbane/10/2007 Florida/4/2006 Season Dose Volp p p Grp (μg) (mL) Titer Enhanc value Titer Enhanc value Titer Enhancvalue 1 15 0.5 320 — — 320 — — 279 — — 2 15 0.3 1280 4.0 0.011 844 2.60.023 557 2.0 0.059 3 5 0.3 1470 13.8 0.010 1114 10.4 0.032 970 10.40.089 4 5 0.3 1470 13.8 0.002 1470 13.8 0.002 422 4.5 0.002 5 5 0.1 9709.1 0.069 640 6.0 0.006 640 6.9 0.031 Overall 5-15 — — 10.2 — — 8.2 6.0Enhancement over TIV = (test group mean/group 1 mean) × (H Ag dose ofgroup 1/H Ag dose of test group) Student's T test in each case iscompared to group 1 Fluzone positive control

Immunopharmacokinetcis: Antibody response kinetics were similar for eachof the three component strains, as shown in FIGS. 4A, B, and C. MAS-1enhanced titers remained elevated throughout days 56, 70, and 84 for all3 viral strains, indicating that the adjuvanted vaccine of the inventionadministered by IM vaccination extends the period for maintainingprotective HAI titers over those induced by the standard 15 μg/H antigendose of Fluzone.

EXAMPLE 6

Safety/Toxicology Evaluations: The scope of the rabbitimmunopharmacology included: observations of animal general well-beingduring the in vivo phase, and formal evaluations of safety andtoxicology, including animal weights, necropsy, visual inspection andorgan weights under veterinary supervision, and histopathologyassessments of the 20 selected organs in Table 7 from Groups 1 (Fluzone15 μg), Group 2 (MAS-1/TIV 15 μg), and Group 9 (MAS-1 placebo),respectively; were performed in compliance with GLP. In addition, allinjection sites were examined post mortem by visual and histologicalassessments on all rabbits.

TABLE 7 Tissues collected at necropsy for histopathology from eachrabbit in Groups 1, 2 and 9. Sample Tissue Collected Type Heart, Spleen,Adrenal, Ovary, Popliteal lymph node, Whole Mandibular lymph node, BrainKidney, Liver, Lung, Pancreas, Aorta, Stomach, Duodenum, SectionJejunum, Ileum, Cecum, Colon, Esophagus, Trachea

General Safety: Fluzone TIV and all MAS-1/TIV formulations appeared safeand well tolerated during the in vivo phase of the study assessed byindependent observations made by animal welfare personnel. No animals atany adjuvanted vaccine dose or Fluzone showed signs of ill health at anypoint during the study. No statistically significant differences in bodyweights taken on day 72 were observed between the adjuvanted vaccineGroups 2-8 or MAS-1 placebo Group 9, or Fluzone Group 1.

Necropsy: All rabbits in each of Groups 1, 2, and 9 were necropsied andthe panel of 20 organs harvested in compliance with GLP. Three minorabnormalities were found, including two in the Fluzone group 1 (loosestool) and one in MAS-1 placebo group 9 (small lobe of extra-splenictissue). None of these abnormalities were considered to be related toany of the test materials, nor were they believed to have any bearing onthe study outcome. Eleven of the 20 organs collected from each rabbit ingroups 1, 2 and 9 were weighed prior to fixation. The organ weights, theorgan to body weight ratios, and the organ to brain weight ratios werecompared between the three groups. No statistically significantdifferences between the groups were found in these comparisons, withthree exceptions: First: the mandibular lymph nodes, which are distal tothe injection sites in the rear legs, were heavier in group 1 than ingroups 2 or 9 rabbits. Second: the right (but not left) popliteal lymphnodes were slightly larger in group 9 than in group 2 rabbits. Third:Brain/body weight ratio was statistically smaller in group 2 than group9 rabbits. The actual brain weights between groups 2 and 9 were notdifferent and apparent differences in brain/body weight ratio can beattributed to the differences in overall body weights between Groups 2and 9. None of these observations were considered to be significant tothe safety of any of the test articles.

Histopathology: Histopathology on the 20 organs collected from groups 1,2, and 9 rabbits was performed in compliance with GLP. Occasionalinstances of cellular infiltrates and/or congestion noted for someorgans were concluded to be typical background findings. This studyconfirmed the expected lack of systemic toxicity and found no evidenceof histomorphologic differences between rabbits treated with theadjuvanted vaccine of the invention at 15 μg/H antigen and rabbitstreated with either Fluzone or MAS-1 placebo.

Injection site tolerance: Immediately after sacrifice of the rabbits onstudy day 84, injection sites were scored visually (macroscopic) andbiopsy specimens collected. The biopsies were subsequently studied forhistopathology and graded by a Board Certified veterinary pathologist.Visual and histology evaluations are presented in Table 8.

TABLE 8 Visual and Histology Scores at sites 1 and 2 after IM injectionin rabbit thigh muscle Injection site 1 (Day 0 Inj) Injection site 2(Day 28 Inj) Vaccine Dose Vol Visual Histology Visual Histology Grp TIVμg/H mL Mean Range Mean Range Mean Range Mean Range 1 Fluzone 15.0 0.5 00 0 0 0 0 0 0 2 MAS-1/TIV 15.0^(a) 0.3 0 0 1.5   0-2.5 0.4 0-1 0.5  0-1.5 3 MAS-1/TIV 5.0^(b) 0.3 0 0 0 0 0.3 0-1 1.5   0-2.5 4 MAS-1/TIV5.0^(a) 0.3 0.1 0-0.5 0 0 0.7 0-1 1.4 0-3 5 MAS-1/TIV 5.0^(c) 0.1 0 0 00 0.1   0-0.5 1.0 0-3 6 MAS-1/TIV 1.67^(a) 0.3 0 0 0 0 0.5 0-1 1.5 0-3 7MAS-1/TIV 1.67^(a) 0.1 0 0 0 0 0.4 0-1 1.1 0-3 8 MAS-1/TIV 0.56^(b) 0.30.2 0-0.5 0 0 0.3 0-1 0.7 0-2 9 Placebo 0 0.3 0 0 0.2 0-1 0.1   0-0.50.7 0-1 ^(a)Emulsified directly to the indicated concentration of Hantigen. ^(b)Emulsion prepared at strength for Group 2 was diluted 1:3with placebo emulsion ^(c)Same emulsion as that used for Group 2

Key to visual pathology scores Key to histolopathology scores Normaltissue 0-0.5 Normal tissue or very mild 0-0.5 inflammation Minimalpathology 1-1.5 Mild active or residual chronic 1-1.5 pathology Moderatepathology 2-2.5 Moderate active chronic 2-2.5 inflammation 3 Severechronic inflammation/ 3 pathology

Visual injection site scores of ≦1, and histology scores of ≦2 areindicative of a tolerable formulation; some inflammation is anticipatedand correlates with the immune response enhanced by the adjuvant.Fluzone was well tolerated at injection sites 1 and 2, but consistentwith its lower immune response, only limited inflammation was seenhistologically at both sites. MAS-1/TIV was well tolerated both visuallyand histologically at the site of the first injection. Of the 35MAS-1/TIV injection sites examined, three (one in Group 4, two in Group8) received visual scores of 0.5, indicating barely discernabledifference between the injection site and surrounding muscle tissue. Theremaining 32 MAS-1/TIV and all 5 Fluzone and 5 MAS-1placebo firstinjection sites appeared normal. The second MAS-1/TIV injection was morereactogenic, with 7/35 sites at 0.5, 10/35 sites at 1.0 and 18/35 with 0visual scores. The second MAS-1 placebo injection had 1/5 sites at 0.5with 4/5 at a 0 visual score. All second injection sites with Fluzonehad 0 visual score.

The macroscopic injection site scores were generally supported byhistological examinations of biopsy specimens. Thus, histopathology wasnot observed at the first injection site in any rabbits except for thosein group 2, where moderate microscopic reactions were noted in tworabbits and mild reactions seen in two others. Increased inflammationwas found at the second injection site for all MAS-1/TIV formulations,with scores ranging from 0 to 3. Mild inflammation at site two was notedin two rabbits receiving MAS-1 placebo, while Fluzone did not elicitinflammation at the second injection sites.

Both visual and histopathology assessments support a single injectionregimen anticipated for MAS-1 adjuvanted TIV to be administered IM as aprophylactic influenza vaccine in humans at doses of 15 μg/H antigen orless.

EXAMPLE 7

Preparation of POU Vaccine Formulation from Commercial SeasonalInfluenza Vaccine Supplies.

Preparation of the vaccine miniemulsions of the invention requiresmixing and emulsification the aqueous phase containing the proteinantigen with oily vehicle. Standard stock influenza vaccine contains 30μg/mL (TIV 30) of each H antigen or TIV containing higherconcentrations, (a 4-fold higher strength seasonal TIV vaccine for theelderly comprising 120 μg/mL (TIV 120) Sanofi Pasteur) may be used foreach H antigen. The vaccines of the invention are preferably about a30:70 (w/w) water-in-oil emulsion. To produce POU vaccine formulationsof the invention for clinical purposes so as to provide doses at 1.0,3.0, and 5.0±15% μg/H antigen, the TIV 30 and TIV 120 vaccines arecombined with MAS-1 vehicle as indicated in the schematic shown in Table9.

POU Process Outline:

Step 1: In each case, for 1, 3 and 5 μg/H antigen doses in MAS-1formulation, 0.5 mL of Fluzone TIV 30 and TIV 120 are removed from theFluzone vials according to the schema A and B shown in Table 9.

Step 2; The TIV aqueous phase solutions are then transferred byinjection into single use, pre-filled sterile PBS vials and mixed byhand.

Step 3: In each case, 0.5 mL of each [diluted] aqueous phase is thentransferred by injection into single use pre-filled, sterile vialscontaining 1.2 g of MAS-1 adjuvant. The vial contents are mixed byshaking vigorously for 30 seconds to produce a milky pre-emulsion.

Step 4: The aqueous and MAS-1 pre-emulsion mixture is transferred into a2.0 ml syringe and emulsified using the double syringe method.

TABLE 9 POU formulations at 1, 3, and 5 ± 15% μg/H antigen for clinicalpurposes MAS-1/TIV Step Dose μg 1.0 μg/H 3.0 μg/H 5.0 μg/H A1 TIV 30 mL0.5 0.5 0.5 A2 PBS for dilution mL 1.0 — — A3 Diluted Aq. Phase mL 0.50.5 0.5 MAS-1 g 1.2 1.2 1.2 A4 MAS-1/TIV yield mL 1.9 1.9 1.9 Dose VolmL 0.3 0.3 0.5 B1 TIV 120 mL 0.5 0.5 0.5 B2 PBS for dilution mL 1.67 0.23 — B3 Diluted Aq. Phase mL 0.5 0.5 0.5 MAS-1 g 1.2 1.2 1.2 B4MAS-1/TIV yield mL 1.9 1.9 1.9 Dose vol mL 0.1 0.1  0.12

Based on the results in rabbits (Table 6A) showing statisticalequivalency between as low as 0.56 μg/H antigen in MAS-1/TIV and 15 μg/Hantigen in standard TIV, we anticipate that from about 1 to 5 μg/Hantigen dose in formulated in MAS-1 delivered in either 0.1 mL or 0.3 mLshould be optimal for use in the elderly human patients.

POU Syringe Hand Mixing Process: At 1 to 2 mL scale, the mixingprocedure takes 90-120 seconds using a pre-set number of cycles. Thegeometry of the syringe method and flow characteristics are critical tosuccessful emulsification. The emulsion pre-mix is drawn into a 2 mLNorm-Ject syringe and then attached to a 3-way stopcock. A second 2 mLsyringe is then attached to the stopcock at 90°. The assembly is claspedfirmly around the 3-way stopcock. The pre-emulsion is passed from onesyringe to the second by carefully depressing the first syringe plungerwith the palm of the other hand. This constitutes 1 pass or cycle. Fullemulsification is then achieved by completing 125 cycles within 90-120(or 150 seconds at 5 mL scale). (Syringes—2 to 5 mL Norm-Ject, sterile,single-use, all plastic Tuberculin, Air-tite Products Co. Inc, VA USA;Henke Sass Wolf GMBH, Tuttlingen, Germany; Kruuse UK Ltd, UK; Syringeneedles—18 or 21 gauge, sterile, single use; 3-way stopcock, lipidresistant, Vygon Corp, catalog number 876.00)

Typically the emulsions are expelled into a clean sterile vial and canbe transferred from the pharmacy to the patient area prior to removingthe prescribed injection volume. The POU method is effective forproducing from 1 to 5 mL of adjuvanted vaccine that should remain stablefor at least 24 hours at ambient temperature (Tables 3 and 4). The lowviscosity, free flowing emulsions enable accurate low volume dosing withas little as 0.1 to 0.3 mL injection volumes. This means that multiple(from 3 to 10 at 1 mL scale to 15 to 50 at 5 mL scale) POU doses of theadjuvanted influenza vaccine of the invention can be simply and quicklyprovided by this POU method, particularly useful in the event of apandemic influenza outbreak.

The POU process is useful in epidemic emergency response situationswhere there is a need for a potent adjuvant system that can beformulated and administered with antigens that are in short supply. Theoil component of the emulsion can be stockpiled with kits such asdescribed above, comprising syringes, vials, stopcocks, etc. anddistributed independently of the required vaccine antigen, which cansubsequently be delivered as it becomes available. This type of POUsystem can be particularly useful for a rapid response in epidemic andbiodefense situations where there is very short time period between theoutbreak of the infectious agent and the identification of an effectivetarget antigen and its production in sufficient quantity for vaccinationof large populations.

A kit useful for the point-of-use administration of a water-in-oilemulsion vaccine against an infectious agent comprises a sterile vial ofan adjuvant oil, a sterile vial of aqueous PBS for combining with aninfectious agent antigen, two sterile syringes, a lipid resistantthree-way stopcock, a 21 gauge sterile needle, a 25 gauge sterile needleand a sterile vial for storing the formulated water-in-oil emulsionvaccine. The adjuvant oil should be useful with a wide range of proteinantigens. In one embodiment of the invention the adjuvant oil comprisesmannide monooleate, squalene and squalane. Other oil adjuvants may beformulated as described above. The MAS-1 adjuvant available from MerciaPharma, Inc. may be used as the adjuvant oil for the vaccines of theinvention. Other oil adjuvants such as the Montanide adjuvants availablefrom SEPPIC, SA, Paris, France, that are not mineral oil based but arecomprised of animal or vegetable sourced oils may also be used toformulate vaccines of the invention according to the methods describedherein. The kits may be used with a wide range of influenza antigens orantigens of other infectious agents or combinations of such antigens,including toxins derived from said pathogens.

1. A vaccine composition comprising a nanoparticulate water-in-oilemulsion wherein the aqueous phase of the emulsion comprises from about25% to about 35% by weight of the composition and contains an influenzaantigen, and wherein the oil phase of the emulsion comprises squaleneand squalane, a first emulsifier which is mannide monooleate or sorbitolmonooleate and a second emulsifier selected from the group consisting ofpolyoxyl-40-hydrogenated castor oil, sorbitan monopalmitate, apolysorbate, Hypermer B239 and Hypermer B246, and wherein the medianaqueous globule size is from about 0.3 μm to about 1 μm.
 2. Thecomposition of claim 1 wherein the first emulsifier is mannidemonooleate and the second emulsifier is polyoxyl-40-hydrogenated castoroil.
 3. The composition of claim 1, wherein metabolizable oils comprisefrom about 85% to about 90% by weight of the oil.
 4. The composition ofclaim 1, wherein said first emulsifier comprises from about 9% to about12% by weight of the oil.
 5. The composition of claim 1, wherein saidsecond emulsifier comprises from about 0.5% to about 0.7% by weight ofthe oil.
 6. The composition of claim 1, wherein said squalene comprisesa range from about 40% -about 60% by weight.
 7. The composition of claim1, wherein said squalane comprises a range from about 40%-about 60% byweight.
 8. The composition of claim 6 wherein said squalene and saidsqualane comprise a ratio from about 50% squalene to about 50% squalaneby weight.
 9. The composition of claim 1, wherein said nanoparticulatewater-in-oil emulsion comprises an adjuvant oil vehicle from about65%-about 75% by weight.
 10. The composition of claim 1 wherein saidnanoparticulate water-in-oil emulsion vaccine comprises an adjuvant oilvehicle.
 11. The composition of claim 1, wherein said nanoparticulatewater-in-oil emulsion vaccine comprises an adjuvant oil vehicle fromabout 65%-about 75% by weight.
 12. The composition of claim 1, whereinsaid nanoparticulate water-in-oil emulsion vaccine comprises an aqueousphase further comprising a protein antigen.
 13. The composition of claim1, wherein said nanoparticulate water-in-oil emulsion vaccine comprisesan aqueous phase from about 25%-about 35% by weight.
 14. The compositionof 1, wherein said nanoparticulate water in oil emulsion vaccinecomprises an aqueous phase from about 27%-about 33% by weight.
 15. Thecomposition of claim 1, wherein said water in oil emulsion vaccinecomprises said antigen having a diameter from about 10 nM to about 1micron.
 16. The composition of claim 1, wherein said water in oilemulsion vaccine comprises said antigen having a median diameter ofabout 300 nM.
 17. The composition of claim 1, wherein said water in oilemulsion vaccine is used in conjunction with a protein solubilizer. 18.The composition claim 1, wherein said protein solubilizer is urea orDMSO.
 19. A kit for the point-of-use administration of a nanoparticulatewater-in-oil emulsion vaccine against an infectious agent comprising: avial of an adjuvant oil comprising mannide monooleate, squalene andsqualane, a vial of aqueous PBS for combining with an antigen, at leastone syringe, a lipid resistant three-way stopcock, at least one needle,and a vial for storing the formulated water-in-oil emulsion vaccine. 20.The kit of claim 19, wherein metabolizable oils comprise from about 85%to about 90% by weight of the oil.
 21. The kit of claim 19, wherein afirst emulsifier comprises from about 9% to about 12% by weight of theoil.
 22. The kit of claim 19, wherein a second emulsifier comprises fromabout 0.5% to about 0.7% by weight of the oil.
 23. The kit of claim 19,wherein said squalene comprises a range from about 40%-about 60% byweight.
 24. The kit of claim 19, wherein said squalane comprises a rangefrom about 40%-about 60% by weight.
 25. The kit of claim 24 wherein saidsqualene and said squalane comprise a ratio from about 50% squalene toabout 50% squalane by weight.
 26. The kit of claim 19, wherein saidnanoparticulate water-in-oil emulsion comprises an adjuvant oil vehiclefrom about 65%-about 75% by weight.
 27. The kit of claim 19, whereinsaid nanoparticulate water-in-oil emulsion vaccine comprises an adjuvantoil vehicle.
 28. The kit of claim 19, wherein said nanoparticulatewater-in-oil emulsion vaccine comprises an adjuvant oil vehicle fromabout 65%-about 75% by weight.
 29. The kit of claim 19, wherein saidnanoparticulate water-in-oil emulsion vaccine comprises an aqueous phasefurther comprising a protein antigen.
 30. The kit of claim 19, whereinsaid nanoparticulate water-in-oil emulsion vaccine comprises an aqueousphase from about 25% -about 35% by weight.
 31. The kit of claim 19,wherein said nanoparticulate water in oil emulsion vaccine comprises anaqueous phase from about 27% -about 33% by weight.
 32. The kit of claim19, wherein said water in oil emulsion vaccine is used in conjunctionwith a protein solubilizer.
 33. The kit of claim 19, wherein saidprotein solubilizer is Urea or DMSO.
 34. A method for a point of useformulation of an adjuvanted nanoparticulate water-in-oil emulsionvaccine comprising: preparing an aqueous solution of a proteinaceousantigen, transferring a suitable amount of the aqueous solution to asterile container containing a predetermined amount of an adjuvant oilcomprising mannide monooleate, squalene and squalane, shaking thecontainer so as to mix the contents to form a milky pre-emulsion,transferring an aliquot of the milky pre-emulsion to a first sterilesyringe, connecting the first sterile syringe to a sterile three-waystopcock, attaching a second sterile syringe to the three-way stopcockat a 90 degree angle to the first syringe, passing the emulsion from onesyringe to the other by manually depressing the plunger of eachrespective syringe in a serial fashion for a sufficient number of cyclesso as to create an emulsion of the aqueous solution in the adjuvant oilwherein the median globule size is from about 0.3 μm to about 1 μm. 35.A kit of claim 19, wherein said kit is for the point-of-useadministration of a nanoparticulate water-in-oil emulsion vaccineagainst an infectious agent comprising: a vial of an adjuvant oilcomprising mannide monooleate, squalene and squalane, a vial of aqueousPBS for combining with an antigen, two syringes, a lipid resistantthree-way stopcock, two needles and a vial for storing the formulatedwater-in-oil emulsion vaccine.